PCR protocols and conditions

materials and solutions

• Generuler mix DNA ladder Fermentas/Lab Force AG price:565CHF
25 x 50µg 50-100µl aliquots store @-20˚C
preparation: 100µl ladder+ 100µl 6xloading buffer + 400µl mQH2O
• 1kb+ DNA ladder Invitrogen Cat# 10787-026 price:893.50CHF
20-50µl aliquots store @-20˚C
preparation: 50µl ladder + 450µl 2x loading buffer
• Proteinase K Merck/VWR Cat# 1.24568.0500 price:417CHF
1ml aliquots store @-20˚C
• dNTPs 20mM Promega/Catalys price:1729CHF
Cat# U1410 200µmol (2 x 100µlmol each)
100µl aliquots store @-20˚C
• 10X PCR buffer GeneAMP® Applied Biosystems price:312CHF
1Pack =150ml Part No. N808-189
1.5ml aliquots store @-20˚C
• Taq: home made 50µl aliquots. Store at -80˚C until use.
Store at -20˚C after first thawing.
• Oligos: Invitrogen
Resuspend to 200mM (stock solution) in TrisHCl 10mM pH8. 50µl aliquots
Working solution:20mM. 1/10 dilution in mQH2O from stock solution. 100µl aliquots
Store at -80˚C until use. Store at -20˚C after first thawing.

• Usual PCR mix preparation in 25µl final volume

Preparation for 1 sample
mQH2O 21µl
10X PCR buffer 2.5µl
dNTPs (20mM) 0.25µl
Oligos 1 and 2 0.25µl each
Taq 0.025µl
FLP Shh∆
Primers: FLPfor-FLPR Primers: Shh1930-Shh1931 wt allele
Shh1931-Shh1840 mut allele

PCR program PCR program:

94˚C 5’ 94˚C 5’
94˚C 30” 94˚C 1’
60˚C 30” x30cycles 60˚C 30” x30cycles
72˚C 1’ 72˚C 1’
72˚C 5’ 1.5% agarose gel
72˚C 5’ 1.5% agarose gel
Generuler ladder mix
10˚C ∞ Generuler ladder mix 10˚C ∞

FLP for: CTA ATG TTG TGG GAA ATT GGA GC
FLP R: CTC GAG GAT AAC TTG TTT ATT GC
Gre
Primers: GRU1-GRL1 wt allele
ßGal(U3014)-Neo(21) ∆allele
Shh1931: GGA CAC CAT TCT ATG CAG GG
Shh1930: GAA GAG ATC AAG GCA AGC TCT GGC
Shh1840: ATG CTG GCT CGC CTG GCT GTG GAA
XtJ
Primers:XtJF-XtJR wt allele
XtJ580F-XtJ580R mut allele

PCR program PCR program:

94˚C 5’ 94˚C 5’
94˚C 30” 94˚C 1’
62˚C 30” x40cycles 60˚C 30” x30cycles
72˚C 2’ 72˚C 1’
72˚C 5’ 72˚C 5’
10˚C ∞ 1.5% agarose gel
1kb+ ladder
10˚C ∞ 1.5% agarose gel
Generuler ladder mix

GRU1: ATG AAT CGC ACC GCA TAC ACT G XtJF: GGG TGA ACA GCA TCA AAA TGG AG
GRL1: TCC AAG TCG ATG GAT ATG CAA CG XtJR: ATA GCC ATG TGG TGG TGC CCA TG
ßGal(U3014): GGC ACA TGG CTG AAT ATC GAC GG XtJ580F: TAC CCC AGC AGG AGA CTC AGA TTA G
Neo(21): AAG CGC CTC CCC TAC CCG GTA XtJ580R: AAA CCC GTG GCT CAG AGC AAG
Bmp4lox/∆ Hoxb7GFP
Primers: BMP4U1-BMP4L1-wt and flox alleles Primers: Hoxb7s-Hoxb7as
BMP4U1-3’ßGal -∆ allele

PCR Program: PCR Program:

94˚C 5’ 94˚C 5’
94˚C 30” 94˚C 30”
58˚C 30” x40cycles 60˚C 1’20” x30cycles
72˚C 1’ 72˚C 1’15”
72˚C 5’ 72˚C 5’
10˚C ∞ 10˚C ∞
1.5% agarose gel 1.5% agarose gel
Generuler ladder mix 1kb+ ladder

BMP4U1:GTG TGT GTA GGG TGT GAG GGA GAA A Hoxb7s:AGC GCG ATC ACA TGG TCC TG
BMP4L1:CCC GGT CTC AGG TAT CAA ACT AGC A Hoxb7as:ACG ATC CTG AGA CTT CCA CAC T
3’ßGal:CCC AGT CAC GAC GTT GTA AAA CGA C

ßactinSTOP GFP CRE

Primers: ActinF-ActinR Primers: CRE1-CRE2

PCR Program: PCR Program:

94˚C 5’ 94˚C 5’
94˚C 30” 94˚C 30”
61˚C 30” x30cycles 61˚C 30” x35cycles
72˚C 30” 72˚C 30”
72˚C 5’ 1.5% agarose gel 72˚C 5’ 1.5% agarose gel
Generuler ladder mix
10˚C ∞ 10˚C ∞ Generuler ladder mix

ActinF:CAT GAA GCA GCA CGA CTT CT CRE1:GCC TGC ATT ACC GGT CGA TGC AAC GA
ActinR:GCT TGT CGG CCA TGA TAT AG CRE2:GTG GCA GAT GGC GCG GCA ACA CCA TT
LoxP
Primers: Ex24U-Ex24L wt allele
Ex24U-Hygro1 loxP allele

PCR Program:
94˚C 5’
94˚C 30”
58˚C 30” x40cycles
72˚C 1’30”
72˚C 5’
10˚C ∞
1.5% agarose gel
1kb+ ladder

Ex24U: GAA AGA CTG CTG CAG AAG GAA GC

EX24L: TCC AAA GAA GGC ACA GGG GAC TT
Hygro1: GGG TGG GGT GGG CAT TAG ATA AA